Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases

When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.

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Publication Details

Journal
Nature Biomedical Engineering
Published
2024-11-04
DOI
https://doi.org/10.1038/s41551-024-01277-5
Citations
33
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
7.72

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article

Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases

Omer Habib, Segi Kim, Gue‐Ho Hwang, Heon Seok Kim et al.
33 citations
Nature Biomedical Engineering
CRISPR and Genetic Engineering
7.72
article

Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases

Omer Habib, Segi Kim, Gue‐Ho Hwang, Heon Seok Kim, Seok‐Hoon Lee, Chan Hyuk Kim, Sangsu Bae, Hyeon‐Ki Jang, Sun Kim, Youngkuk Kim, Minsik Oh
article en
33 citations

Abstract

When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.

Nature Biomedical EngineeringVol. 9(1)
Seoul National University (KR), Korea Advanced Institute of Science and Technology (KR), Kangwon National University (KR), Cancer Research Institute (US), Seoul National University Hospital (KR), National University College (PR), Institute of Molecular Biology (AM), Hanyang University (KR), Myongji University (KR)
National Research Foundation, Seoul National University, National Research Foundation of Korea, Iran Telecommunication Research Center, Institute for Information and Communications Technology Promotion
Zero hunger
Openalex Percentile: Top 2%
CRISPR and Genetic Engineering
7.72
DOIOpen Access PDFOpenAlex
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